KR20190044986A - Pharmaceutical composition for use in preventing or treating osteoporosis containing betaine as an active ingredient - Google Patents
Pharmaceutical composition for use in preventing or treating osteoporosis containing betaine as an active ingredient Download PDFInfo
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- KR20190044986A KR20190044986A KR1020170137707A KR20170137707A KR20190044986A KR 20190044986 A KR20190044986 A KR 20190044986A KR 1020170137707 A KR1020170137707 A KR 1020170137707A KR 20170137707 A KR20170137707 A KR 20170137707A KR 20190044986 A KR20190044986 A KR 20190044986A
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- South Korea
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- pharmaceutical composition
- betaine
- formula
- compound represented
- pharmaceutically acceptable
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/205—Amine addition salts of organic acids; Inner quaternary ammonium salts, e.g. betaine, carnitine
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
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- Food Science & Technology (AREA)
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- Pharmacology & Pharmacy (AREA)
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
Description
본 발명은 베타인 또는 이의 약학적으로 허용 가능한 유도체를 유효성분으로 함유하는 골다공증의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis comprising betaine or a pharmaceutically acceptable derivative thereof as an active ingredient.
뼈는 체내 구조와 골격계를 구성하고, 그 중 골 조직은 무기질로 구성된 세포외기질, 새로운 골 형성을 하는 조골세포(osteoblast), 골 흡수를 하는 파골세포(osteoclast), 골세포 등 여러 종류의 세포들로 구성되어 있으며, 이들이 서로 균형을 이루면서 무기질화 과정이 끊임없이 일어나는 복잡하고 활동적인 조직이다. 정상적인 성인에서는 파골세포 활성과 조골세포 활성이 균형을 이루어 항상성이 유지되며, 뼈는 성장이 끝난 후 일정한 주기로 오래된 골을 제거하고 새로운 골로 채우는 과정인 골 재형성을 진행한다. 이러한 골 재형성은 체내의 국소적 인자와 호르몬의 상호작용에 의해 조절되며, 조골세포의 활성도가 낮아져 골 형성이 감소되거나 파골세포의 활성도가 강해져 골 흡수가 증가 되는 등의 조골세포와 파골세포간의 활성 불균형은 조직 내 화학조성에는 큰 변화가 없지만 골 질량이 감소하여 골 대사질환을 초래한다.The bone constitutes the internal structure and the skeletal system, and the bone tissue is composed of various types of cells such as an extracellular matrix composed of minerals, osteoblasts forming new bone, osteoclasts performing osteoclast, Are complex and active organizations in which the processes of mineralization are constantly occurring in balance with each other. In normal adults, osteoclast activity and osteoblast activity are balanced to maintain homeostasis. Bones undergo bone regeneration, a process of removing old bone at regular intervals and filling with new bone after growth. Such bone remodeling is regulated by the interaction of local factors and hormones in the body, resulting in decreased osteoblast activity resulting in decreased osteogenesis, increased osteoclast activity, and increased bone resorption. The active imbalance has no significant change in the chemical composition of the tissue, but it decreases bone mass and results in bone metabolic disease.
골 대사 질환 중에서 골다공증(osteoporosis)은 유전적 요인이나 식이, 생활 방식과 같은 환경적 요인에 의해 영향을 받는 복합적인 질병으로 현대사회의 급속한 고령화로 인해 사회적, 의학적 관심에서 사회문제로까지 부상하고 있다. Among osteoporosis diseases, osteoporosis is a complex disease that is affected by environmental factors such as genetic factors, diet and lifestyle, and it is emerging from social and medical concerns to social problems due to rapid aging of modern society .
골 항상성에 의해 인체 하중의 지탱, 기관보호, 조혈작용의 역할을 수행하지만, 각종 질병이나 노화, 흡연, 스트레스와 폐경기 여성들의 급격한 에스트로겐 저하 등의 여러 인자로 인하여 균형이 깨지게 되면 골의 형성보다 골 흡수가 증가하게 되어 결국 골 소실이 일어나게 된다.However, when the balance is broken due to various factors such as various diseases, aging, smoking, stress and sudden estrogen depression of postmenopausal women, bone formation is more likely to be caused by osteoporosis than osteoporosis Increased absorption leads to bone loss.
골다공증의 치료방법으로 현재까지는 칼슘보충제, 비타민 D 대사 산물과 티아자이드 이뇨제(thiazide diuretic) 요법, 칼시토닌(calcitonin) 요법, 비스포스포네이트(bisphosphonate) 요법, 에스트로겐(estrogen) 요법 등이 이용되고 있다.To date, calcium supplementation, vitamin D metabolites and thiazide diuretic therapy, calcitonin therapy, bisphosphonate therapy, and estrogen therapy have been used to treat osteoporosis.
그러나 폐경성 골다공증(postmenopausal osteoporosis)의 치료로 사용하는 에스트로겐을 장기간 사용할 경우에 오심, 체중증가, 불규칙한 자궁출혈, 자궁내막암 및 유방암과 같은 부작용을 유발하는 것으로 보고되고 있으며, 아직까지 일반적인 골다공증 치료방법 중에서 골 형성을 촉진하여 골 소실을 완전하게 회복시킬 수 있는 치료방법은 나오지 않고 있다. 특히 기존 치료법의 대부분을 차지하고 있는 파골세포 활성억제를 통해 골 흡수를 억제하는 골다공증 치료법들은 근본적으로 골다공증을 치료할 수 있는 치료법이 아니기 때문에 완치에 한계가 있으며 실질적인 골밀도 증가 및 골 강화가 이루어질 수 있는 조골세포 활성의 증가가 필수적으로 요구된다. However, long-term use of estrogen for the treatment of postmenopausal osteoporosis has been reported to cause adverse effects such as nausea, weight gain, irregular uterine bleeding, endometrial cancer and breast cancer, There is no treatment method that can completely restore bone loss by promoting osteogenesis. In particular, osteoporosis treatments that inhibit osteoclast activity by inhibiting osteoclast activity, which is a major part of conventional treatments, are fundamentally not treatable to treat osteoporosis. Therefore, osteoclasts An increase in activity is indispensably required.
또한, 골다공증은 약물의 단기 투여만으로는 치료할 수 없고 약물의 장기 투여가 필수적인 질환이므로, 약물을 장기 투여할 때에도 부작용이 없고 에스트로겐을 대체할 수 있을 만큼 우수한 약효를 갖는 새로운 물질들의 개발이 요구되고 있다.In addition, since osteoporosis can not be treated only by short-term administration of a drug and long-term administration of the drug is essential, development of new substances having a drug efficacy sufficient to replace estrogen without side effects is required for prolonged administration of the drug.
이에 따라, 최근에는 기존 치료법의 부작용 최소화와 골 소실을 최소화하면서 골 형성을 촉진할 수 있는 한약재 및 식품 등의 천연물 유래 활성성분을 이용한 대체요법 연구가 활발히 진행되고 있다. 일례로, 매실 추출물로부터 분리된 화합물들을 이용한 조골세포 분화유도 및 파골세포 분화억제 효과를 나타내는 골다공증 예방 또는 치료용 조성물이 연구 및 개발된바 있으며(특허문헌 1), 엉겅퀴 추출물을 이소플라본과 혼합하여 효능을 상승시킨 골다공증 치료 및 예방용 조성물에 대한 연구도 이루어진 바 있다(특허문헌 2).Recently, alternative therapies using active ingredients derived from natural materials such as herbal medicines and foods that can promote bone formation while minimizing side effects of existing therapies and minimizing bone loss have been actively studied. For example, a composition for the prevention or treatment of osteoporosis, which exhibits osteoblast differentiation induction and osteoclast differentiation inhibitory effects using compounds isolated from plum extract, has been studied and developed (Patent Document 1), and the thistle extract is mixed with isoflavone Studies on compositions for treating and preventing osteoporosis with increased efficacy have been made (Patent Document 2).
한편, 베타인(Betaine)은 메틸기를 세개 가진 아미노산으로 동·식물계에 널리 존재하고, 특히 해양 무척추동물, 시금치, 구기자, 무 등에 다량 함유되어 있어 음식을 통해 공급받거나 생체 내에서 탈수소효소(dehydrogenase)와 베타인 알데히드 탈수소효소(betaine aldehyde dehydrogenase)에 의한 산화과정을 통해 생성되며, 체내에서 콜린(choline)의 최종 대사 산화물로 간과 신장에서 발현하는 베타인-호모시스테인 메틸트랜스퍼라제(BHMT:betaine-homocysteine methyltransferase)와 메싸이오닌 합성효소(MS: methionine synthase)에 의해 호모시스테인(homocysteine)에 메틸기를 전달하여 메싸이오닌(methionine)을 생성하기 위한 메틸 그룹의 공여체로 작용한다.On the other hand, Betaine is an amino acid having three methyl groups and is widely present in plants and plants. Especially, it is contained in marine invertebrate, spinach, goji, radish and the like and is supplied through food or dehydrogenase in vivo. (Betaine-homocysteine methyltransferase (BHMT), expressed in the liver and kidney as the final metabolite of choline in the body, produced through the oxidation of betaine-aldehyde dehydrogenase ) And methionine synthase (MS), which acts as a donor of the methyl group to generate methionine by transferring the methyl group to homocysteine.
또한, 베타인(Betaine)은 황함유 아미노산 대사를 변화시켜 메싸이오닌(methionine), 시스테인(cysteine)을 황전환작용 경로(TS:transsulfuration pathway)에 의해 최종적으로 타우린(taurine)과 글루타티온(glutathione)을 생성하여 간독성에 보호효과를 나타낸다고 보고된 바 있다. 또 다른 보고에 의하여 고농도의 혈중 호모시스테인은 동맥경화 같은 질병을 일으키는데 이때 충분한 양의 베타인(Betaine)을 공급해 주면 혈중 호모시스테인의 농도가 저하되어 동맥경화를 예방할 수 있을 것으로 예상된다. 이 밖에도 베타인에 관한 여러 선행 연구 결과들이 있지만 베타인이 조골세포의 증식 및 분화에 미치는 영향에 대한 연구결과는 아직 보고된 바 없다.In addition, Betaine changes the metabolism of sulfur-containing amino acids so that methionine and cysteine are ultimately converted to taurine and glutathione by the transsulfuration pathway (TS) And thus has a protective effect on hepatotoxicity. According to another report, a high concentration of homocysteine causes a disease such as arteriosclerosis. If a sufficient amount of betaine is supplied, the concentration of homocysteine in the blood will be lowered and it is expected to prevent atherosclerosis. In addition, there are several previous studies on betaine, but no study has been reported on the effect of betain on proliferation and differentiation of osteoclasts.
본 발명자들은 근본적으로 골다공증을 치료하기 위해 실질적인 골밀도 증가 및 골 강화를 위해 조골세포의 활성을 증가시키고, 장기간 복용하여도 부작용이 적은 새로운 약학적 조성물을 개발하기 위해 노력하던 중, 베타인 또는 이의 약학적으로 허용 가능한 유도체들이 조골세포의 증식 및 분화를 촉진함을 확인함으로써 본 발명을 완성하였다.The inventors of the present invention have been intensively working to develop a new pharmaceutical composition which is effective for increasing osteoporosis and for increasing osteoblast activity for increasing bone density and strengthening bones, The present inventors have accomplished the present invention by confirming that allowable derivatives promote the proliferation and differentiation of osteoblasts.
따라서, 본 발명의 목적은 골다공증 예방 및 치료 효능이 우수하고 장기간 복용하여도 부작용이 적은 약학적 조성물 및 건강기능식품을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition and a health functional food having excellent osteoporosis prevention and treatment efficacy and having little side effects even when taken for a long time.
상기 목적을 달성하기 위해,In order to achieve the above object,
본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체를 유효성분으로 함유하는 골다공증의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating osteoporosis, which comprises a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
상기 화학식 1로 표시되는 화합물은 베타인(Betaine)으로 명명될 수 있다.The compound represented by the formula (1) may be named betaine.
상기 약학적 조성물은 조골세포 분화를 촉진시키는 것을 특징으로 할 수 있다. The pharmaceutical composition may be characterized in promoting osteoblast differentiation.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 1 mM 인 것을 특징으로 할 수 있다.The concentration of the compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof may be 0.01 to 1 mM.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체는 Runx2(runt-related transcription factor 2), Id1(inhibitor of DNA binding 1), Dlx5(distal-less homeobox 5), OC(osteocalcin) 및 ALP(alkaline phosphatase) 중 어느 하나 이상의 조골세포 분화 마커 유전자의 발현량을 증가시키는 것을 특징으로 할 수 있다.The compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof may be selected from Runx-related transcription factor 2 (Runx2), inhibitor of DNA binding 1, Dlx5 (distal-less homeobox 5), osteocalcin and an alkaline phosphatase (osteoclast differentiation marker gene).
상기 약학적 조성물은 산제, 과립제, 정제, 경질 캡슐제, 연질 캐슐제 또는 주사제의 형태로 제형화 되는 것을 특징으로 할 수 있다.The pharmaceutical composition may be formulated in the form of powders, granules, tablets, hard capsules, soft capsules or injections.
또한, 본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체를 유효성분으로 함유하는 골다공증의 예방 또는 개선용 건강기능식품을 제공한다.The present invention also provides a health functional food for preventing or ameliorating osteoporosis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
[화학식 1][Chemical Formula 1]
상기 화학식 1로 표시되는 화합물은 베타인(Betaine)으로 명명될 수 있다.The compound represented by the formula (1) may be named betaine.
상기 건강기능식품은 조골세포 분화를 촉진시키는 것을 특징으로 할 수 있다.The health functional food may be characterized by promoting osteoblast differentiation.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 1 mM 인 것을 특징으로 할 수 있다.The concentration of the compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof may be 0.01 to 1 mM.
본 발명에 따른 약학적 조성물은 조골세포 분화 마커 유전자인 Id1, Dlx5, Runx2, ALP, OC의 발현량을 증가시키고, 조골세포의 석회화를 증가시킴으로써 조골세포의 분화를 촉진하는 효과가 있어 골다공증의 예방 또는 치료용 의약품 또는 건강식품으로 널리 이용될 수 있다.The pharmaceutical composition according to the present invention increases the expression level of the osteoblast differentiation marker genes Id1, Dlx5, Runx2, ALP, and OC and increases osteoblast calcification, thereby promoting osteoblast differentiation. Thus, osteoporosis prevention Or therapeutic medicines or health foods.
또한, 파골세포를 표적으로 하기보다는 조골세포의 활성을 증가시킴으로써 골형성을 촉진 시켜 근본적으로 골다공증을 치료할 수 있는 장점이 있다.In addition, rather than targeting osteoclasts, osteoblast activity can be increased to promote osteogenesis, thereby osteoporosis can be fundamentally treated.
도 1은 베타인(Betaine)의 농도에 따른 MTT 분석의 결과 그래프이다.
도 2는 베타인(Betaine)에 의한 조골세포 분화 마커의 발현 정도를 비교한 <실험예 2> 에 따른 결과이다.
도 3은 베타인(Betaine)이 조골세포의 석회화 결절 형성능에 미치는 영향을 확인하기 위한 <실험예 3> 에 따른 결과이다.1 is a graph of MTT analysis according to the concentration of betaine.
FIG. 2 shows results of Experimental Example 2 comparing the expression level of osteoblast differentiation markers by Betaine.
FIG. 3 shows the results of Experiment 3 for confirming the effect of betaine on the ability of osteoblast to form calcified nodules.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 명세서 및 청구범위에 사용되는 용어나 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 아니되며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다.The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary meanings and the inventor may properly define the concept of the term to describe its invention in the best possible way It should be construed as meaning and concept consistent with the technical idea of the present invention.
본 발명은 골다공증의 예방 또는 치료용 약학적 조성물 및 건강기능식품에 관한 것이다.The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating osteoporosis.
본 명세서에서 용어 골다공증은 뼈의 양이 감소하고 질적인 변화로 인하여 뼈의 강도가 약해진 상태를 의미하고, 골다공증의 예방, 개선 및 치료라 함은 골 밀도 저하, 골 손실로 인한 각종 질병을 모두 포함하는 것으로 해석된다.As used herein, the term osteoporosis refers to a state in which the amount of bone is reduced and the strength of the bone is weakened due to a qualitative change, and the prevention, improvement and treatment of osteoporosis includes all kinds of diseases caused by decreased bone density and bone loss .
본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체를 유효성분으로 함유하는 골다공증의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating osteoporosis, which comprises a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
[화학식 1][Chemical Formula 1]
상기 화학식 1로 표시되는 화합물은 베타인(Betaine)으로 명명될 수 있다.The compound represented by the formula (1) may be named betaine.
상기 화학식 1로 표시되는 화합물은 무수물, 수화물, 또는 염산염이나 그 외 물에 용해 시켰을 때 상기 화학식 1로 표시되는 화합물을 유리할 수 있는 다른 종류의 약학적으로 허용 가능한 염을 포함한다. The compound represented by the general formula (1) includes an anhydride, a hydrate, or another pharmaceutically acceptable salt which may be useful as a hydrochloride salt or a compound represented by the general formula (1) when dissolved in water.
상기 약학적으로 허용 가능한 염이란 통상적인 방법으로 제조한 염을 의미하며, 당업자에게 공지된 방법으로 제한없이 사용될 수 있다. 약학적으로 허용 가능한 염은 약리학적 또는 생리학적으로 허용되는 다음 무기산과 유기산 및 염기로부터 유도된 염을 포함하지만 이에 한정되는 것은 아니다. 적합한 산의 예로는 염산, 브롬산, 황산, 질산, 과염소산, 푸마르산, 말레산, 인산, 글리콜산, 락트산, 살리실산, 숙신산, 톨루엔-p-설폰산, 타르타르산, 아세트산, 시트르산, 메탄설폰산, 포름산, 벤조산, 말론산, 나프탈렌-2-설폰산, 벤조설폰산등을 들 수 있다. 적합한 염기로부터 유도된 염은 알칼리 금속(예를들어 나트륨), 알칼리토금속(예를들어 마그네슘, 암모늄)등을 포함할 수 있다.The pharmaceutically acceptable salt means a salt prepared by a conventional method and can be used without limitation in a method known to a person skilled in the art. Pharmaceutically acceptable salts include, but are not limited to, those derived from the following inorganic and organic acids and bases which are pharmacologically or physiologically acceptable. Suitable acids include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, , Benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzosulfonic acid, and the like. Salts derived from suitable bases may include alkali metals (e.g., sodium), alkaline earth metals (e.g., magnesium, ammonium), and the like.
상기 화학식 1로 표시되는 화합물인 베타인은 공지된 방법에 의해 천연물로부터 추출된 것이나 합성된 것을 사용할 수 있으며, 그 제조방법이나 기원은 한정되지 않는다.The betaine, which is a compound represented by the above formula (1), can be extracted from natural products or synthesized by a known method, and the production method and origin are not limited.
베타인은 수산물에 널리 분포하며, 어류에는 보통 0.1% 이하로 존재하나 무척추동물인 오징어, 문어, 새우 등의 근육에는 어류의 수배에 이르게 존재하며 멍게에도 존재한다. 식물계에는 맥아, 버섯류, 과실 특히 명화주과에 많이 포함되어 있다. Betaine is widely distributed in aquatic products. It is usually present in fishes at less than 0.1%, but exists in the muscles of squid, octopus, and shrimp, which are invertebrates, at the worms of fishes. In the vegetable field, malt, mushroom, and fruit, especially, are included in the master page.
또한, 베타인은 감칠맛 성분으로 지방산의 담즙산 분비촉진, 간세포 재생 촉진 및 손상 예방, 독성 물질 배출 및 해독 작용, 저혈압 개선 기능 및 혈압 안정에 효능이 있다고 알려져 있다. 베타인 염산염은 예로부터 위액의 산도를 조정하는 의약품으로 사용되어 왔으며, 이외에도 동물의 성장촉진, 지방대사, 간 기능의 정상화에 좋은 영향을 끼치는 것으로 알려져 있다.Betaine is also known to have a beneficial effect on the promotion of bile acid secretion of fatty acids, the promotion of hepatocyte regeneration and damage prevention, toxic substance release and detoxification, hypotension, and blood pressure stability. Betaine hydrochloride has long since been used as a medicinal product to regulate the acidity of gastric juices and is known to have a good effect on the growth of animals, fat metabolism and normalization of liver function.
상기 화학식 1로 표시되는 화합물의 약학적으로 허용 가능한 유도체는 특별히 제한된 것은 아니나, 감마-부티로베타인(gamma-butyrobetaine), 타우로베타인(taurobetaine), 카르니틴(carnitine) 등이 있을 수 있다.The pharmaceutically acceptable derivatives of the compound represented by Formula 1 include, but are not limited to, gamma-butyrobetaine, taurobetaine, carnitine, and the like.
상기 약학적 조성물은 조골세포 분화를 촉진시키는 것을 특징으로 할 수 있다. The pharmaceutical composition may be characterized in promoting osteoblast differentiation.
조골세포의 분화를 촉진시키는 화합물은 골다공증 및 골절 등과 같은 질환 및 증상의 예방 또는 치료에 사용될 수 있을 뿐만 아니라, 골 강화를 목적으로 하는 치주질환 치료에도 사용될 수 있으며(Zhang et al., ShanghaiKou Qiang Yi Xue 7(2), pp99-103, 1998; Cahill et al, Biol Blood Marrow Transplant 10(10), pp709-717, 2004), 화상으로 비롯되는 골 성장 장애 치료에 유용하게 사용될 수 있다는 연구도 보고된바 있다(Klein et al.,Osteoporos Int. 16(6), pp631-635, 2005).Compounds that promote osteoclast differentiation can be used not only for the prevention or treatment of diseases and symptoms such as osteoporosis and fracture but also for the treatment of periodontal disease for the purpose of bone strengthening (Zhang et al., Shanghai Kou Qiang Yi It has also been reported that it may be useful for the treatment of bone growth disorders resulting from burns, as reported by Xue 7 (2), pp 99-103, 1998; Cahill et al, Biol Blood Marrow Transplant 10 (10), pp709-717, (Klein et al., Osteoporos Int. 16 (6), pp. 631-635, 2005).
상기 약학적 조성물은 조골세포 등의 부족으로 인한 골 형성에 문제가 있는 질환에 제한 없이 사용될 수 있으며, 구체적으로 에스트로겐의 부족으로 인한 인터루킨-1(IL-1)에 의한 골 파괴와 염증성 질환 등이 포함될 수 있고, 과도한 파골 세포의 골 흡수에 의한 골다공증, 뼈전이암 병소(bone metastatic lesion), 원발성으로 뼈에 생성된 종양, 류마티스성 또는 퇴행성 관절염, 치주질환, 염증성 치조골 흡수질환, 염증성 뼈 흡수 질환 및 파제트병(Paget's disease) 등과 같은 병리학적 골 질환으로 골 파괴를 촉진하는 질환이 포함될 수 있다.The pharmaceutical composition can be used without limitation in diseases causing bone formation due to deficiency of osteoblasts and the like. More specifically, the pharmaceutical composition can be used for the treatment of bone destruction and inflammatory diseases caused by interleukin-1 (IL-1) And can be used to treat osteoporosis, bone metastatic lesion, bone-derived tumors, rheumatoid or degenerative arthritis, periodontal disease, inflammatory alveolar bone disease, inflammatory bone resorption disease due to excessive osteoclast bone resorption And Paget's < Desc /
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 1 mM 인 것을 특징으로 할 수 있다.The concentration of the compound represented by
보다 상세하게는, 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 1 mM일 수 있고, 바람직하게는 0.1 내지 1 mM일 수 있으며, 더욱 바람직하게는 0.5 내지 1 mM일 수 있다.More specifically, the concentration of the compound represented by
만일 농도가 0.01 mM 미만일 경우 약학적인 효과가 미미하게 나타날 수 있고, 1 mM를 초과할 경우 세포독성이 나타날 수 있는 문제점이 있다.If the concentration is less than 0.01 mM, the pharmacological effect may be insignificant. If the concentration is more than 1 mM, cytotoxicity may occur.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체는 Runx2(runt-related transcription factor 2), Id1(inhibitor of DNA binding 1), Dlx5(distal-less homeobox 5), OC(osteocalcin) 및 ALP(alkaline phosphatase) 중 어느 하나 이상의 조골세포 분화 마커 유전자의 발현량을 증가시키는 것을 특징으로 할 수 있다.The compound represented by
조골세포 분화는 호르몬, 뼈형성단백질(bone morphogenetic proteins:BMPs)과 같은 사이토카인, Runx2(runt-related transcription factor 2), Id1(inhibitor of DNA binding 1), Dlx5(distal-less homeobox 5), OC(osteocalcin), ALP(alkaline phosphatase)과 같은 다양한 전사인자에 의해서 조절된다. 그 중에서도 Id1, Dlx5, Runx2는 조골세포 분화 초기에 발현되는 필수적인 유전자로 이들 유전자의 발현은 조골세포 분화가 가속화됨을 의미한다.Osteoblast differentiation can be induced by a combination of hormones, cytokines such as bone morphogenetic proteins (BMPs), runt-related transcription factor 2 (Runx2), inhibitor of DNA binding 1, Dlx5 (distal-less homeobox 5) (osteocalcin), and alkaline phosphatase (ALP). Among them, Id1, Dlx5, and Runx2 are essential genes that are expressed at the early stage of osteoblast differentiation. Expression of these genes means that osteoblast differentiation is accelerated.
구체적으로, Runx2(runt-related transcription factor 2)는 조골세포 분화에 있어 중요한 조절자로 많이 알려져 있으며 다분화능세포 또는 조골전구세포에서 ALP(alkaline phosphatase), OC(osteocalcin) 그리고 골격시알로단백질(bone sialoprotein)과 같은 유전자를 조절함으로써 조골세포 분화를 촉진하는 유전자이다.Runx-related transcription factor 2 (Runx2) is known to be an important regulator of osteoblast differentiation, and it is known that ALP (alkaline phosphatase), OC (osteocalcin) and bone sialoprotein ) To control osteoblast differentiation.
Id1(inhibitor of DNA binding 1)은 조골세포에서 세포의 성장과 분화 사이의 균형에 영향을 미치는 전사인자이고, 조골세포 특이적 분화에 있어서 초기 단계에 중요한 역할을 하는 것으로 알려져 있다. 뿐만 아니라 근원세포, 중간엽 줄기세포에서 조골세포 분화로 전환될 때 Id1(inhibitor of DNA binding 1) 유전자 발현의 조절에 따라 분화가 촉진된다. Id1 (inhibitor of DNA binding 1) is a transcription factor that affects the balance between cell growth and differentiation in osteoblasts and is known to play an important role in the early stages of osteoblast-specific differentiation. In addition, differentiation is promoted by the regulation of Id1 (inhibitor of DNA binding 1) gene expression when transformed into osteoblast differentiation from myoblast and mesenchymal stem cells.
Dlx5(distal-less homeobox 5) 또한 뼈에 의해 유도되며 Runx2(runt-related transcription factor 2)의 프로모터에 직접적으로 결합하여 전사를 조절하고, 조골세포 분화의 후기 표지 유전자인 OC(osteocalcin)의 유전자 발현을 조절함으로써 조골세포 분화를 조절한다.Dlx5 (distal-less homeobox 5) is also induced by bone and directly binds to the promoter of Runx2 (runt-related transcription factor 2) to regulate transcription, and gene expression of OC (osteocalcin), the late marker gene of osteoblast differentiation To control osteoblast differentiation.
상기 약학적 조성물은 임상 투여시에 경구 또는 비경구로 투여가 가능하며, 일반적인 의약품 제제의 형태로 사용될 수 있다.The pharmaceutical composition can be administered orally or parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation.
상기 약학적 조성물은 산제, 과립제, 정제, 경질 캡슐제, 연질 캐슐제 또는 주사제의 형태로 제형화 되는 것을 특징으로 할 수 있다.The pharmaceutical composition may be formulated in the form of powders, granules, tablets, hard capsules, soft capsules or injections.
보다 상세하게는, 각각 통상적인 방법에 따라 산제, 과립제, 정제, 캡슐, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 멸균 주사용액, 사전 충전식 주사 용액제의 형태 또는 동결건조된 형태로 제형화할 수 있으나, 이에 제한되는 것은 아니다.More specifically, the pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppository sterilized injection solutions, But the present invention is not limited thereto.
제형화할 경우에는 통상 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 제조될 수 있다.In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.
경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 상기 유효 성분에 적어도 하나 이상의 부형제, 예컨대, 전분, 칼슘 카르보네이트, 수크로오스, 락토오스, 젤라틴 등을 혼합하여 제조할 수 있다. 또한, 단순한 부형제 이외에도 마그네슘 스테아레이트, 탈크와 같은 윤활제도 사용될 수 있다.Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.
경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 통상적으로 사용되는 단순 희석제인 물, 액체 파라핀 이외에 다양한 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 함께 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 좌제 등이 포함된다.Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. may be included in addition to water and liquid paraffin which are simple diluents commonly used have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.
본 발명의 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 증상의 정도, 약물 형태, 투여 경로 및 기간에 따라 적절하게 선택될 수 있다.A preferable dosage of the pharmaceutical composition of the present invention can be appropriately selected depending on the condition and the weight of the patient, the degree of symptoms, the drug form, the administration route and the period.
또한, 본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체를 유효성분으로 함유하는 골다공증의 예방 또는 개선용 건강기능식품을 제공한다.The present invention also provides a health functional food for preventing or ameliorating osteoporosis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
[화학식 1][Chemical Formula 1]
상기 화학식 1로 표시되는 화합물은 베타인(Betaine)으로 명명될 수 있다.The compound represented by the formula (1) may be named betaine.
상기 건강기능식품은 조골세포 분화를 촉진시키는 것을 특징으로 할 수 있다.The health functional food may be characterized by promoting osteoblast differentiation.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 1 mM 인 것을 특징으로 할 수 있다.The concentration of the compound represented by
보다 상세하게는, 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 1 mM일 수 있고, 바람직하게는 0.1 내지 1 mM일 수 있으며, 더욱 바람직하게는 0.5 내지 1 mM일 수 있다.More specifically, the concentration of the compound represented by
만일 농도가 0.01 mM 미만일 경우 약학적인 효과가 미미하게 나타날 수 있고, 1 mM를 초과할 경우 세포독성이 나타날 수 있는 문제점이 있다.If the concentration is less than 0.01 mM, the pharmacological effect may be insignificant. If the concentration is more than 1 mM, cytotoxicity may occur.
상기 건강기능식품은, 비제한적으로 각종 음료, 껌, 차, 과자, 비타민 복합체, 건강 보조식품 등의 형태로 제조될 수 있다.The health functional food may be manufactured in the form of, but not limited to, various drinks, gum, tea, confectionery, vitamin complex, health supplement, and the like.
또한, 상기 건강기능식품은 골다공증 개선을 목적으로 건강식품에 첨가되는 경우도 포함하며, 식품의 종류에 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.In addition, the health functional food includes a case where it is added to health food for the purpose of improving osteoporosis, and there is no particular limitation on the kind of food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.
상기 건강기능식품의 바람직한 섭취량은 섭취자의 상태 및 체중, 증상의 정도, 식품 형태, 섭취 기간에 따라 다르며 적절하게 선택될 수 있다.The preferred amount of the health functional food to be consumed depends on the condition and the weight of the recipient, the degree of symptoms, the type of food, the period of consumption, and may be appropriately selected.
뼈의 조직은 세포외기질과 조골세포, 파골세포, 골세포 등 여러 종류의 세포들로 구성되어 있으며 뼈의 재형성이 계속적으로 일어난다. 뼈의 재형성은 파골세포에 의한 골 흡수와 조골세포에 의한 골 형성이 균형을 이루는 과정으로 진행된다. Bone tissue consists of various types of cells including extracellular matrix, osteoblast, osteoclast, osteocyte, and bone resurfacing. Bone resurfacing is a process that balances osteoclast-induced bone resorption and osteoblastic bone formation.
골의 생성과정은 기질의 증식, 성숙, 석회화의 세 단계로 이루어진다. 골 형성을 하는 조골세포는 미분화 간엽 세포에서 유래된 전조골세포(preosteoblasts)가 뼈 표면에 도달하여 성숙한 조골세포로 분화되고, 10-20% 정도는 골세포로 되어 석회화 조직에 묻히는 것으로 알려져있다. 따라서 조골세포가 석회화 과정에 중요한 역할을 한다는 것을 알 수 있다. The process of bone formation consists of three stages: proliferation of the substrate, maturation, and calcification. It is known that osteoblastic osteoblast cells are derived from undifferentiated mesenchymal cells, and preosteoblasts reach the bone surface and differentiate into mature osteoblasts. About 10-20% of the bone cells are buried in calcified tissues. Therefore, it can be seen that osteoblasts play an important role in the calcification process.
MC3T3-E1 전조골세포(preosteoblasts)는 골기질의 축적, 석회화, 성장요소들에 대한 효과, 형태와 대사의 변화 등을 연구하는데 이용되고 있으며, 아스코르부산(Ascobric acid)이 공급된 배지에서 MC3T3-E1 전조골세포(preosteoblasts)는 단계에 따라 조골세포 형질의 발현 및 석회화 세포외기질 형성을 나타낸다. 발달단계에서 초기 4-10일간은 증식기, 10-16일은 골 기질 형성 및 성숙기, 16-30일은 석회화기로 석회화기에서는 결절 수가 증가 되어 비타민 K 의존성 칼슘 결합 단백질인 오스테오칼신(OC)이 높게 나타나는 것으로 알려져 있다.MC3T3-E1 proosteoblasts are used to study bone accumulation, calcification, effects on growth factors, changes in morphology and metabolism, and in MC3T3- E1 preosteoblasts represent the expression of osteoblastic traits and the formation of calcified extracellular matrix in stages. It is known that osteocalcin (OC), which is a vitamin K-dependent calcium binding protein, is highly expressed in the developmental stage during the initial 4-10 days, during bone formation and maturation in 10-16 days, during calcification in 16-30 days, have.
본 발명에서는 골조직에 존재하는 조골세포와 유사한 MC3T3-E1 전조골세포(preosteoblasts)를 이용하여 베타인이 조골세포의 증식 및 분화와 석회화 결절 형성능에 미치는 영향을 알아보기 위한 실험을 진행하였으며, 그 결과 베타인은 조골세포의 증식 및 분화와 석회화 결절형성에 긍정적인 효과를 나타냄을 확인할 수 있었다.In the present invention, an experiment was conducted to examine the effect of betain on the proliferation and differentiation of osteoblasts and the ability to form calcified nodules using MC3T3-E1 preosteoblasts similar to osteoblasts present in bone tissue. As a result, Showed positive effects on the proliferation and differentiation of osteoblasts and formation of calcified nodules.
이하, 본 발명을 구체적으로 설명하기 위해 실시예 및 실험예를 들어 상세하게 설명하기로 한다. 그러나 본 발명에 따른 실시예 및 실험예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 아래에서 상술하는 실시예 및 실험예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해서 제공되는 것이다.Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, and thus are not limitative of the present invention, It is provided for a complete description.
<< 실험예Experimental Example 1> 베타인( 1> Betain ( BetaineBetaine )이 조골세포 증식에 미치는 영향 확인) On osteoclast proliferation
1-1. 실험방법1-1. Experimental Method
(1) 실험재료 및 세포배양(1) Experimental materials and cell culture
먼저, 실험에 사용되는 재료로 베타인 하이드로클로라이드(Sigma-Aldrich Co., St. Louis, MO, USA)를 멸균수에 녹여 사용하였고, 조골세포(osteoblasts)는 마우스 유래의 MC3T3-E1 세포(ATCC, Manassas, VA, USA)를 사용하였으며, 세포배양에는 알파-최소필수영양배지(alpha-minimum essential medium:α-MEM)(Sigma-Aldrich Co., St. Louis, MO, USA)와 포스페이트 버퍼드 살린(Phophate Buffered Saline; PBS)(Gibco, GrandIsland, NY, USA)을 사용하였다.First, osteoblasts were used as mouse-derived MC3T3-E1 cells (ATCC (Sigma-Aldrich Co., St. Louis, Mo., USA) (Sigma-Aldrich Co., St. Louis, Mo., USA) and phosphate buffered saline (PBS) were used for cell culture. Phosphate Buffered Saline (PBS) (Gibco, Grand Island, NY, USA) was used.
MC3T3-E1 조골세포는 페니실린 및 스트렙토마이신(Gibco)이 함유된 1% 항균 용액(antibacterial-antifungal solution)과 10% 가열불활화 소태아혈청(heat-inactivated fetal bovine serum: FBS)(ATLAS Biologicals, Fort collins)을 함유한 α-MEM 배지로 배양하여 PBS로 세척한 후 0.25% 트립신 에틸렌다이아민테트라아세트산(EDTA)(Gibco) 용액을 사용해 5% CO2, 37 ℃ 이하의 인큐베이터 환경에서 배양하고, 2일에 한번 씩 배양액을 교체하여 실험에 사용하였다. 실험조건으로 MC3T3-E1 전조골세포를 베타인의 농도를 달리하여 배양하였고, MC3T3-E1 전조골세포에 일반 배지를 사용한 것(대조군)과 MC3T3-E1 전조골세포에 베타인을 포함한 배지를 사용한 것(실험군)을 배양하여 사용하였다.MC3T3-E1 osteoblast cells were treated with 1% antibacterial-antifungal solution containing penicillin and streptomycin (Gibco) and 10% heat-inactivated fetal bovine serum (FBS) (ATLAS Biologicals, Fort collins), washed with PBS, and cultured in an incubator environment of 5% CO 2 and 37 ° C or less using 0.25% trypsin ethylenediaminetetraacetic acid (EDTA) (Gibco) solution. 2 The culture medium was replaced once per day and used in the experiment. As the experimental condition, MC3T3-E1 precursor bone cells were cultured at different betaine concentrations, and MC3T3-E1 precursor bone cells were used as control medium (control group) and MC3T3-E1 precursor bone cells were used as media containing betaine Experimental group) was used for culture.
(2) 세포독성실험(2) Cytotoxicity test
시료의 세포독성을 측정하기 위하여 3-(4,5-디메틸-티아졸-2-일)-2,5-디페닐테트라졸리움-브로마이드(MTT)(Sigma-Aldrich Co., St. Louis, MO, USA)를 사용한 MTT assay로 살아있는 세포의 생존율을 측정하였다.(MTT) (Sigma-Aldrich Co., St. Louis, Mo.) was used to determine the cytotoxicity of the sample. , USA) was used to measure the survival rate of living cells.
48-웰 플레이트 중 12 웰에 MC3T3-E1 전조골세포(preosteoblasts)를 2x104 세포/웰의 농도로 분주하고 24시간 후에 베타인을 농도별(0, 0.1, 0.5, 1, 5, 25 mM)로 투여하여 24시간 동안 배양하였다. 배양 후 5 mg/ ml 농도의 MTT 염색약을 10 배 희석하여 각 웰에 300 μl씩 첨가한 뒤 2시간 동안 37 ℃ 인큐베이터에서 반응시켰다. 시간 경과 후 배지를 제거하고 각 웰에 디메틸설폭사이드(DMSO)를 100 μl씩 첨가하여 불용성의 포마잔(formazan) 결정을 용해시키고 540 nm에서 마이크로플레이트 리더를 이용하여 흡광도를 측정하여 세포 생존율을 확인하였다.MC3T3-E1 preosteoblasts were dispensed in 12 wells of a 48-well plate at a concentration of 2 x 10 4 cells / well, and after 24 hours, betaine was added at concentrations of 0, 0.1, 0.5, 1, 5, 25 mM And cultured for 24 hours. After the incubation, MTT dye at a concentration of 5 mg / ml was diluted 10-fold and 300 μl was added to each well, followed by incubation for 2 hours in a 37 ° C incubator. After the passage of time, the medium was removed and 100 μl of dimethylsulfoxide (DMSO) was added to each well to dissolve the insoluble formazan crystals. The absorbance was measured at 540 nm using a microplate reader to confirm cell viability Respectively.
1-2. 실험결과1-2. Experiment result
상기 실험의 결과를 도 1에 나타내었다. 나타낸 바와 같이 MC3T3-E1 전조골세포(preosteoblasts)는 베타인 처리 시 세포 증식률이 증가하는 경향을 보였고 0.5 mM 에서 10% 정도의 증식률을 보였으며, 특히 1 mM 에서 100% 이상의 세포 생존율을 나타내 높은 증식률을 보였다. 반면, 비교적 고농도인 5 mM으로 처리시 70%의 세포 생존율을 나타내 대조군에 비해 세포 생존율이 감소하는 세포독성을 나타냈으며, 특히 25 mM에서 15%의 세포 생존율을 나타내었다. 따라서 조골세포의 증식을 저해하지 않는 베타인의 최적 작용 농도를 1 mM으로 결정하고 다음 실험을 진행하였다.The results of the above experiment are shown in Fig. As shown, the MC3T3-E1 preosteoblasts showed a tendency to increase cell proliferation at the time of treatment with betaine and showed a proliferation rate of 0.5 mM to 10%, especially at 1 mM, a high cell proliferation rate Respectively. On the other hand, cell viability was decreased by 70% at 5 mM, and cell viability was 15% at 25 mM. Therefore, the optimal concentration of betaine that does not inhibit the proliferation of osteoblast cells was determined to be 1 mM and the following experiment was conducted.
<< 실험예Experimental Example 2> 베타인(Betaine) 처리에 따른 조골세포 분화 2> Osteoblast differentiation by Betaine treatment 마커의Marker 발현 분석 Expression analysis
2-1. 실험방법2-1. Experimental Method
(1) RNA 추출 및 cDNA 합성(1) RNA extraction and cDNA synthesis
RNA 추출을 위해 MC3T3-E1 전조골세포(preosteoblasts)를 계대배양한 6-웰 플레이트의 배양액(media)을 석션(suction)한 후, 각 웰에 트리졸 용액(Bioscience technology) 300 μl를 처리하고 스크랩퍼(scrapper)로 세포를 모아 마이크로-튜브에 옮겨 5분간 반응시켰다. For RNA extraction, media of 6-well plates in which MC3T3-E1 preosteoblasts were subcultured was treated with 300 μl of a solution of triosol (Bioscience technology) in each well, The cells were collected in a scrapper and transferred to a micro-tube for reaction for 5 minutes.
이어서, 클로로폼 60 μl를 첨가하여 15초간 와류(vortexing)하고 다시 5분간 반응시켜 원심분리(14500 rpm, 4 ℃, 15 min) 한 후 상층액의 RNA부분 100 μl를 새로운 마이크로-튜브로 옮겨 이소프로필 에탄올 100 μl를 첨가하고 인버팅(inverting)한 후 4 ℃에서 10분간 반응시켰다. Subsequently, 60 μl of chloroform was added, followed by vortexing for 15 seconds, followed by reaction for 5 minutes. After centrifugation (14500 rpm, 4 ° C., 15 min), 100 μl of the RNA portion of the supernatant was transferred to a new micro- 100 μl of propyl ethanol was added, inverted and reacted at 4 ° C for 10 minutes.
이후, 다시 원심분리(14500 rpm, 4 ℃, 10 min)하여 상층액 제거 후 펠렛(RNA)을 70% 에탄올로 워싱하고 또다시 원심분리(13000 rpm, 4 ℃, 10 min)하였다. 상층액의 에탄올을 제거하여 20분간 건조 시키고, 3차 증류수 20 μl에 30분간 녹여 정량하였다. 그 후 cDNA 합성을 위해 50 ℃에서 1시간, 95 ℃에서 5분간 반응시키고 4 ℃까지 온도를 낮추어 보관하였다.Thereafter, the supernatant was removed by centrifugation (14500 rpm, 4 ° C, 10 min) and the pellet (RNA) was washed with 70% ethanol and centrifuged again (13000 rpm, 4 ° C, 10 min). Ethanol in the supernatant was removed, dried for 20 minutes, and dissolved in 20 μl of tertiary distilled water for 30 minutes. Then, for cDNA synthesis, reaction was carried out at 50 ° C for 1 hour and at 95 ° C for 5 minutes, and the temperature was lowered to 4 ° C.
(2) 역전사 중합효소 연쇄반응(Reverse Transcription Polymerase Chain Reaction, RT-PCR)(2) Reverse Transcription Polymerase Chain Reaction (RT-PCR)
cDNA 1 μl, 프라이머(primer) 1 μl, 에메랄드 그린 믹스쳐(emerald green mixture, Takara Bio Inc, Japan) 5 μl, 3차 증류수 3 μl를 혼합한 후, peltier-based thermal cycler(LongGene Scientific Inc Co.,Ltd.)를 이용하여 RT-PCR을 수행하였다. RT-PCR에 사용된 각 프라이머의 시퀀스와 반응 조건을 표 1 및 표 2에 나타내었다.1 μl of the cDNA, 1 μl of the primer, 5 μl of the emerald green mixture (Takara Bio Inc, Japan) and 3 μl of the third distilled water were mixed and then transferred to a peltier-based thermal cycler (LongGene Scientific Inc. , Ltd.) was used for RT-PCR. The sequence of each primer used in RT-PCR and the reaction conditions are shown in Table 1 and Table 2.
그 후 TAE(트리스-아세테이트-EDTA) 버퍼 200 ml, 아가로스(agarose) 2 g, 미도리 그린 어드밴스 DNA 염색 2 μl를 혼합해 녹여 1% 아가로스 겔을 만든 후 전기영동을 실시하고 그 결과로 나온 염색대(band)를 겔 다큐멘테이션시스템(Alpha innotech corporation)을 통해 확인하였다.Then, 200 ml of TAE (Tris-Acetate-EDTA) buffer, 2 g of agarose and 2 μl of Midori Green Advance DNA staining were mixed and dissolved to make 1% agarose gel, and electrophoresis was carried out. Dye band was confirmed by Alpha innotech corporation.
Runx2
Id1
Dlx5
OC
ALP
β-actin
gene
cycle
denaturationPre-
denaturation
(Denaturation)Thermal degeneration
(Denaturation)
(Annealing)Combination
(Annealing)
(Extension)kidney
(Extension)
extensionPost-
extension
5 분95 ℃
5 minutes
30 초95 ℃
30 seconds
30 초62 ° C
30 seconds
30 초
72 ℃
30 seconds
5 분
72 ℃
5 minutes
30 초67 ° C
30 seconds
30 초67 ° C
30 seconds
30 초63 ° C
30 seconds
30 초60 ° C
30 seconds
30 초66 ° C
30 seconds
2-2. 실험결과2-2. Experiment result
24시간(도 2의 A)과 72시간(도 2의 B) 동안 베타인을 처리한 후 회수하여 RNA를 추출한 후, cDNA를 합성하여 RT-PCR을 수행하였다. MTT assay에 따라 세포 독성을 나타내지 않은 1mM의 베타인을 처리한 MC3T3-E1 전조골세포(실험군)와 베타인을 처리하지 않은 MC3T3-E1 전조골세포(대조군)를 비교하여 조골세포 분화 마커인 Runx2(runt-related transcription factor 2), Id1(inhibitor of DNA binding 1), Dlx5(distal-less homeobox 5), OC(osteocalcin), ALP(alkaline phosphatase) 의 발현을 확인하였다.After treating betaine for 24 hours (FIG. 2A) and 72 hours (FIG. 2B), RNA was extracted and cDNA was synthesized and RT-PCR was performed. MC3T3-E1 osteoclast cells (experimental group) treated with 1 mM betaine, which did not show cytotoxicity, and MC3T3-E1 osteoclasts not treated with betaine (control group) and the expression of IL-1, IL-1, IL-1, IL-1, and Dlx5.
확인한 결과, 실험군이 대조군에 비해 조골세포의 분화 마커의 발현이 확연히 증가됨을 알 수 있었다(도 2의 A 참조). As a result, it was found that the expression of osteoblast differentiation markers in the experimental group was significantly increased as compared with the control group (see A in FIG. 2).
또한, 세포독성이 나타나지 않은 베타인의 농도(0.1-1 mM) 중 0.5 mM, 1 mM(실험군)과 베타인을 처리하지 않은 세포(대조군)를 비교한 결과 대조군보다 실험군에서 조골세포 분화 마커의 발현이 증가하였고, 0.5 mM의 베타인을 처리한 조골세포보다 1 mM의 베타인을 처리한 조골세포에서 조골세포 분화 마커의 발현이 증가 하였다(도 2의 B 참조).In addition, the expression of osteoblast differentiation markers in the experimental group was lower than that of the control group in comparison with 0.5 mM, 1 mM (experimental group) and betaine-untreated cells (control group) in the concentration of betaine without cytotoxicity (0.1-1 mM) And the expression of osteoblast differentiation markers was increased in osteoblast treated with 1 mM betaine than osteoblast treated with 0.5 mM betaine (see Fig. 2B).
특히, OC(osteocalcin) mRNA의 발현 정도가 뚜렷하게 증가된 것을 확인할 수 있었는데, 발달단계에서 16일 후 석회화기로 접어들기 때문에 석회화 형성능을 알 수 있으나 2-3일간 베타인을 처리하였을 때도 OC(osteocalcin) mRNA의 발현이 증가된 것으로 보아 베타인이 조골세포의 분화를 촉진시키는 것으로 생각되었다. 반면, 3일째 베타인 처리군에서 ALP(alkaline phosphatase) mRNA의 발현이 줄어들었는데 이는 OC(osteocalcin)이 발현되면 ALP(alkaline phosphatase)의 발현이 감소하기 때문이다.In particular, the expression level of OC (osteocalcin) mRNA was markedly increased. After 16 days in the developmental stage, it became calcified, indicating calcification ability. However, when treated with betaine for 2-3 days, osteocalcin mRNA Was increased, indicating that betaine stimulates osteoblast differentiation. On the other hand, on day 3, the expression of ALP (alkaline phosphatase) mRNA decreased in the betaine-treated group because the expression of osteocalcin (OC) decreased ALP (alkaline phosphatase) expression.
<< 실험예Experimental Example 3> 알리자린 3> Alizarin 레드Red 에스s 염색(Alizarin red s staining) 분석 Analysis of staining (Alizarin red staining)
3-1. 실험방법3-1. Experimental Method
MC3T3-E1 전조골세포(preosteoblasts)를 24-웰 플레이트에 2x104 세포/웰 농도로 분주하고 베타인을 처리한 것과 베타인을 처리하지 않은 것을 분류하여 14일 동안 배양하였다. 배양이 끝난 플레이트는 배양액(media)을 제거하고 70% 에탄올을 이용하여 고정하고 멸균수로 세척한 후 알리자린 레드 용액(Sigma-Aldrich Co., St. Louis, MO, USA)을 첨가하여 실온에서 반응시켰다. 염색된 플레이트를 다시 3차 증류수로 3번 세척한 뒤 염색이 된 것을 확인하였다.MC3T3-E1 dividing the precursor bone cells (preosteoblasts) into a 24-well plate at 2x10 4 cells / well concentration and classification as previously handling the betaine not treated with betaine and were cultured for 14 days. After incubation, the plate was fixed with 70% ethanol, washed with sterile water, and then added with alizarin red solution (Sigma-Aldrich Co., St. Louis, Mo., USA) . The stained plate was washed three times with distilled water three times, and it was confirmed that the stained plate was stained.
3-2. 실험결과3-2. Experiment result
석회화 결절 형성능은 조골세포의 분화에 중요한 바이오 마커로 알리자린 레드 에스 염색으로 확인할 수 있다. 알리자린은 무기질화된 세포 기질의 칼슘을 염색하므로 석회화 결절 형성 정도와 염색 정도가 비례한다. Calcified nodule formation ability is an important biomarker for osteoblast differentiation and can be confirmed by alizarin red staining. Because alizarin stains calcium in mineralized cell matrix, degree of calcification nodule formation and degree of dyeing are proportional.
MC3T3-E1 전조골세포(preosteoblasts)에서 베타인을 처리한 것과 베타인을 처리하지 않은 것을 분류하여 14일 동안 배양한 후 석회화 결절 형성 정도를 관찰하였을 때 베타인을 처리하지 않은 조골세포보다 1 mM의 베타인을 처리한 조골세포에서 석회화 결절 형성 정도가 더 증가되는 것을 확인할 수 있었다(도 3의 A 참조).In the MC3T3-E1 preosteoblasts, those treated with betaine and those without betaine were cultured for 14 days. When the degree of calcified nodule formation was observed, 1 mM betaine was added to the osteoblasts treated with no betaine It was confirmed that the degree of formation of calcified nodules was further increased in the treated osteoblasts (see A in FIG. 3).
또한, MC3T3-E1 전조골세포(preosteoblasts)에 베타인의 농도를 달리하여(0, 1, 2 mM) 14일 동안 배양한 후 석회화 결절 형성 정도를 관찰한 결과 베타인(Betaine)의 농도에 비례하여 석회화 결절 형성 정도가 증가 되는 것을 확인할 수 있었다(도 3의 B참조).In addition, after 14 days of incubation with MC3T3-E1 pre-osteoclasts (0, 1, 2 mM) at different concentrations of betaine, the degree of formation of calcified nodules was observed to be proportional to the concentration of betaine And the degree of formation of calcified nodules was increased (see Fig. 3B).
한편, 본 발명에 따른 상기 화학식 1로 표시되는 화합물은 목적에 따라 여러 형태로 제제화가 가능하다. 하기는 본 발명에 따른 상기 화학식 1로 표시되는 화합물을 활성성분으로 함유시킨 몇몇 제제화 방법을 예시한 것으로 본 발명이 이에 한정되는 것은 아니다.Meanwhile, the compound represented by
<< 제제예Formulation example > >
1-1. 1-1. 산제의Sanje 제조 Produce
화학식 1의 화합물 500 ㎎ 500 mg of the compound of formula (1)
유당 100 ㎎
탈크 10 ㎎ 10 mg of talc
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다. The above components are mixed and filled in airtight bags to prepare powders.
1-2. 정제의 제조1-2. Manufacture of tablets
화학식 1의 화합물 500 ㎎ 500 mg of the compound of formula (1)
옥수수전분 100 ㎎
유당 100 ㎎
스테아린산 마그네슘 2 ㎎ 2 mg of magnesium stearate
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다. After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.
1-3. 캅셀제의 제조1-3. Manufacture of capsules
화학식 1의 화합물 500 ㎎ 500 mg of the compound of formula (1)
옥수수전분 100 ㎎
유당 100 ㎎
스테아린산 마그네슘 2 ㎎2 mg of magnesium stearate
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다. The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.
1-4. 주사제의 제조1-4. Injection preparation
화학식 1의 화합물 500 ㎎500 mg of the compound of formula (1)
주사용 멸균 증류수 적량 Sterile sterilized water for injection
pH 조절제 적량pH adjuster
통상의 주사제의 제조방법에 따라 1 앰플당(2 ㎖) 상기의 성분 함량으로 제조한다. (2 ml) per ampoule in accordance with the usual injection method.
1-5. 액제의 제조1-5. Manufacture of liquid agent
화학식 1의 화합물 100 ㎎ 100 mg of the compound of formula (1)
이성화당 10 g 10 g per isomer
만니톨 5 g 5 g mannitol
정제수 적량Purified water quantity
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬 향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100 ㎖로 조절한 후 갈색 병에 충진하여 멸균시켜 액체를 제조한다.Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, The liquid is prepared by sterilization.
Claims (8)
[화학식 1]
1. A pharmaceutical composition for preventing or treating osteoporosis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
[Chemical Formula 1]
상기 약학적 조성물은 조골세포 분화를 촉진시키는 것을 특징으로 하는 골다공증의 예방 또는 치료용 약학적 조성물.
The method according to claim 1,
A pharmaceutical composition for preventing or treating osteoporosis, wherein the pharmaceutical composition promotes osteoblast differentiation.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 1 mM 인 것을 특징으로 하는 골다공증의 예방 또는 치료용 약학적 조성물.
The method according to claim 1,
The pharmaceutical composition for preventing or treating osteoporosis is characterized in that the concentration of the compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof is 0.01 to 1 mM.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체는 Runx2(runt-related transcription factor 2), Id1(inhibitor of DNA binding 1), Dlx5(distal-less homeobox 5), OC(osteocalcin) 및 ALP(alkaline phosphatase) 중 어느 하나 이상의 조골세포 분화 마커 유전자의 발현량을 증가시키는 것을 특징으로 하는 골다공증의 예방 또는 치료용 약학적 조성물.
The method according to claim 1,
The compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof may be selected from Runx-related transcription factor 2 (Runx2), inhibitor of DNA binding 1, Dlx5 (distal-less homeobox 5), osteocalcin wherein the expression level of the osteoblast differentiation marker gene is at least one of alkaline phosphatase and alkaline phosphatase.
상기 약학적 조성물은 산제, 과립제, 정제, 경질 캡슐제, 연질 캐슐제 또는 주사제의 형태로 제형화 되는 것을 특징으로 하는 약학적 조성물.
The method according to claim 1,
Wherein said pharmaceutical composition is formulated in the form of powders, granules, tablets, hard capsules, soft capsules or injections.
[화학식 1]
A health functional food for preventing or ameliorating osteoporosis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
[Chemical Formula 1]
상기 건강기능식품은 조골세포 분화를 촉진시키는 것을 특징으로 하는 건강기능식품.
The method according to claim 6,
Wherein said health functional food promotes osteoblast differentiation.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 1 mM 인 것을 특징으로 하는 건강기능식품.The method according to claim 6,
Wherein the concentration of the compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof is 0.01 to 1 mM.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115634221A (en) * | 2022-10-21 | 2023-01-24 | 拜澳泰克(沈阳)生物医学集团有限公司 | Application of betaine in regulating mesenchymal stem cell adipogenic and osteogenic differentiation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101318055B1 (en) | 2011-12-13 | 2013-10-14 | 서림바이오 주식회사 | The composition of milk thistle and isoflavones for treatment and prevention of osteoporosis |
| KR101548128B1 (en) | 2013-10-02 | 2015-08-28 | 충남대학교산학협력단 | A composition comprising extract of Japanese apricot or compounds isolated therefrom for preventing or treating osteoporosis |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101318055B1 (en) | 2011-12-13 | 2013-10-14 | 서림바이오 주식회사 | The composition of milk thistle and isoflavones for treatment and prevention of osteoporosis |
| KR101548128B1 (en) | 2013-10-02 | 2015-08-28 | 충남대학교산학협력단 | A composition comprising extract of Japanese apricot or compounds isolated therefrom for preventing or treating osteoporosis |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115634221A (en) * | 2022-10-21 | 2023-01-24 | 拜澳泰克(沈阳)生物医学集团有限公司 | Application of betaine in regulating mesenchymal stem cell adipogenic and osteogenic differentiation |
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